CRISPR-Cas systems are recently discovered, RNA-based immune systems that control invasions of viruses and plasmids in prokaryotes. Prokaryotes with CRISPR-Cas systems capture short invader sequences within the CRISPR loci in their genomes, and small RNAs produced from the CRISPR loci (CRISPR (cr)RNAs) are thought to guide Cas proteins to recognize and silence the invading nucleic acids; however, we know very little about how the key steps in the remarkable CRISPR-Cas immune response pathways occur. In this project, we address the molecular basis for the three steps required for CRISPR-Cas defense through the following specific aims: Determine how CRISPR loci acquire foreign DNA sequences Determine the mechanisms by which functional CRISPR RNAs are produced Delineate the mechanisms of invader silencing Specifically, we aim to obtain a molecular understanding of the CRISPR-Cas defense pathways that function in Streptococcus thermophilus. We will identify the essential cellular machinery (proteins and RNAs) and molecular mechanisms involved in each of the three key phases of CRISPR-Cas defense. We hypothesize that Cas proteins function in each of the major steps in the defense pathway, and we will test this and a number of specific related hypotheses in the proposed studies. The experiments will define the CRISPR-Cas pathways used by multitudes of prokaryotes to survive viral attack. The information gained in this study will contribute directly o ongoing efforts aimed at exploiting CRISPR-Cas systems to both strengthen domesticated bacteria (used to produce safe food, pharmaceuticals and biofuels) and weaken human pathogens and limit the spread of antibiotic resistance.